X-CODE: a dual RNA barcoding system for multi-platform clonal tracking and spatial phenotyping

Rota, R.; Flittner, E.; Tartagni, M.; Bourdim, M.; Westlund, E.; McKenzie, J. S.; Devlin, A.; Thankamony, A.; Pandell, N.; Gabel, F.; Siddiqui, M.; Rosano, D.; Wilkes, A.; Andrew, I.; Choi, K. L.; Game, L.; Gorrini, C.; Magnani, L.; Takats, Z.; Bezzi, M.

doi:10.64898/2026.02.10.705126

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bioRxiv · 10.64898/2026.02.10.705126

X-CODE: a dual RNA barcoding system for multi-platform clonal tracking and spatial phenotyping

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Abstract

Experimental dissection of clonal dynamics in complex tissues requires barcoding systems that are scalable, compatible with different analytical platforms, providing phenotypic and spatial resolution. Here we introduce X-CODE, a dual-expressed RNA barcoding system designed to enable high-complexity clonal tracking across sequencing-based, cytometric, and spatial imaging modalities within a unified experimental framework. X-CODE combines a combinatorial, probe-detectable long RNA barcode with a matched short sequencing barcode, enabling seamless integration of probe-based readouts with sequencing and barcode-guided clonal retrieval. We demonstrate robust X-CODE detection by mass cytometry and imaging-based platforms, including spatial RNA barcode readout using via a repurposed Akoya PhenoCycler-Fusion protocol. In addition, we show compatibility with MALDI mass spectrometry imaging for co-registration of clonal and metabolic information. We further demonstrate the feasibility of X-CODE detection within probe-based spatial transcriptomics using the 10x Genomics Xenium platform. Applied to an in vivo model of androgen deprivation in prostate cancer, X-CODE reveals clonal architecture, selection and clone-specific phenotypic and metabolic plasticity underlying castration resistance. Together, X-CODE provides a flexible and broadly accessible platform for integrated clonal analysis across spatial, phenotypic, and molecular dimensions.

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Rota, R., Flittner, E., Tartagni, M., Bourdim, M., Westlund, E., McKenzie, J. S., Devlin, A., Thankamony, A., Pandell, N., Gabel, F., Siddiqui, M., Rosano, D., Wilkes, A., Andrew, I., Choi, K. L., Game, L., Gorrini, C., Magnani, L., Takats, Z., Bezzi, M.. 2026-02-11. X-CODE: a dual RNA barcoding system for multi-platform clonal tracking and spatial phenotyping. https://doi.org/10.64898/2026.02.10.705126

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